The present invention relates generally to the field of neurobiology. More particularly, the invention relates to the discovery of a new family of evectin proteins and their relation to signal transduction, vesicle trafficking, and diseases associated with aberrations in membrane biosynthesis and organization.
Photoreceptors, oligodendrocytes, and myelinating Schwann cells are highly polarized cells that synthesize an exceptionally large, apically-configured organelle composed almost entirely of extended sheets of tightly compacted and specialized plasma membrane. The compacted disks of the photoreceptor rod outer segments or xe2x80x9cROSxe2x80x9d and the compacted laminae of myelin look remarkably similar and there are no other cell types in the body that maintain the extraordinarily high levels of membrane biosynthesis and maintenance that are characteristic of these cells. These cells regulate the production of plasma membrane, the secretion of proteins (e.g., the proteolipid protein in myelin and rhodopsin in rods), and the localization of lipids and proteins in response to environmental stimuli. (Luini and De Matteis, Trends Cell Biol., 3:290-292 (1993); Bretscher, Cell, 85:465-467 (1996); (Blackshaw and Snyder, J. Neurosci., 17:8074-8082 (1997); Mellman, Annu. Rev. Cell Dev. Biol., 12:575-562 (1996); and Cockcroft, Bioessays, 20:423-432 (1998)).
A differentiating neuron, for example, can organize membrane biosynthesis and protein localization such that its growing axon extends over great distances toward the source of a chemoattractant. (Metin et al., Development, 124:5063-5074 (1997)). Oligodendrocytes of the vertebrate brain elaborate extraordinary quantities of specialized membrane (xcx9c5,000 xcexcm2 per day) in response to molecular cues that trigger their myelination of axons. (Pfeiffer et al., Trends Cell Biol., 3:191-197 (1993)). Defective molecular signaling, which results in aberrant biosynthesis and organization of membrane in these cells, has been associated with human diseases such as Multiple Sclerosis (MS), an autoimmune disease that results in demyelination of CNS axons and oligodendrocyte death. (Ridsdale et al., J Biol Chem. 272:4269 (1997) and Atkins et al., J. Neurochem. 73:1090 (1999).
Vertebrate photoreceptors also achieve similar rates of membrane biosynthesis in the course of assembling and maintaining their outer segments. (Besharse, The Retina: A Model for Cell Biological Studies, Adler and Farber, eds., Academic Press, New York, 1986:297-352). Aberrations in biosynthesis and organization of membrane in cells of the retina have been associated with human diseases such as autosomal dominant familial exudative vitreoretinopathy (adFEVR), an inherited disorder characterized by inflammation of retinal blood vessels, neovascularization and vascular drop-out, generalized hyperpermeability of retinal vessels, and consequent retinal degeneration. (Criswick and Schepens, Am. J. Ophthalmol., 68:578-594 (1969)). For all of the above reasons, it is important to identify molecules that link extracellular signals to changes in membrane biosynthesis and organization.
The discovery of a new family of proteins and nucleic acids encoding these proteins is revealed in this disclosure. This family of molecules is characterized by a structure having a pleckstrin homology domain (PHD), a protein-protein interaction module, a phosphorylation domain, and a hydrophobic domain. The first member of the family discovered was named evectin-1 (xe2x80x9cevt-1xe2x80x9d), from the Latin evectus, meaning carried or moved forward. A second member of the family, which bears 40% total homology to evt-1 but greater homology within regions of the molecule, is named evectin-2 (xe2x80x9cevt-2xe2x80x9d).
Embodiments of the invention include a purified or isolated nucleic acid encoding a polypeptide having a pleckstrin homology domain and a hydrophobic membrane-binding domain. This molecule can also have a nucleic acid sequence encoding a protein binding domain or a nucleic acid sequence encoding a phosphorylation domain. Nucleic acids encoding evectins, evectin polypeptides, and fragments of these molecules are embodiments of the invention. Some embodiments also concern a nucleic acid having a nucleotide sequence selected from the group consisting of: SEQ. ID. NO: 1, SEQ. ID. NO: 2, SEQ. ID NO: 3, SEQ. ID. NO: 4, SEQ. ID. NO: 5, SEQ. ID. NO. 11, and SEQ. ID. NO. 12 or a sequence complementary thereto. Further embodied in this invention are purified or isolated nucleic acid sequences encoding a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.
Other embodiments include purified or isolated polypeptides having a pleckstrin homology domain and a hydrophobic domain. As above, these molecules can further include a protein binding domain and/or a phosphorylation domain. Evectins, evectin polypeptides, and fragments of these molecules are embodiments of the invention. Some polypeptides of the invention also have an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.
Antibodies to evectins are also embodiments. These antibodies can be monoclonal or polyclonal. One example are antibodies capable of specifically binding to a protein comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. Some of these antibodies can specifically bind to a polypeptide comprising at least 10 consecutive amino acids of said protein. Desirable antibodies of the invention are capable of specifically bind to evt-1 protein, but not to evt-2 protein or vice versa (i.e., a purified or isolated antibody capable of specifically binding evt-2 protein, but does not specifically bind evt-1 protein).
Methods of identifying a binding partner that interacts with evt-1 or evt-2 is also an embodiment. By one approach, a support comprising evt-1, evt-2 or a representative fragment thereof is provided; the support is contacted with a candidate binding partner; and a biological complex comprising evt-1 or evt-2; and the candidate binding partner, is detected. The detection of such a complex indicates that said candidate binding partner interacts with evt-1 or evt-2.
A computerized system for identifying an agent that interacts with evt-1 or evt-2 is also an embodiment. One embodiment, for example, includes a first data base comprising protein models of the amino acid sequences as set forth in SEQ. ID. NOS. 6-10; a second data base comprising the composition of a plurality of candidate binding partners; a search program that compares the protein model of the first data base with the compositions of the candidate agents of the second database; and a retrieval program that identifies a candidate binding partner that interacts with the protein model of the first database. In some embodiments, the candidate binding partners are selected from the group consisting of: a peptide, a peptidomimetic, and a chemical. Another related embodiment concerns a computer-based system for identifying a target sequence having homology to an evectin molecule. This system includes a database comprising one of the sequences of SEQ ID NOS: 1-12 or a representative fragment thereof; a search program that compares a target sequence to sequences in the database to identify homologous sequence(s), and a retrieval program that obtains said homologous sequence(s).
A method of identifying an organism in need of treatment or prevention of a defect in vesicle trafficking, signal transduction, G protein binding, or membrane biosynthesis and organization is also an embodiment of the invention. This method is practiced by obtaining a biological sample comprising RNA or protein from an organism; providing a probe that interacts with an evt protein or an RNA encoding an evt protein; contacting the biological sample with the probe under conditions that allow the probe to bind to the evt protein or the probe to bind with the RNA encoding an evt protein in the biological sample; detecting the amount of probe that interacts with the evt protein or the RNA encoding an evt protein in the biological sample so as to determine the concentration or level of expression of the evt protein or the RNA encoding an evt protein; and identifying the organism as an organism in need of treatment or prevention of a defect in vesicle trafficking, signal transduction, G protein binding, or membrane biosynthesis and organization based on the concentration or level of expression of the evt protein or the RNA encoding an evt protein detected in the sample.
Another way to identify an agent that modulates evt-mediated signal transduction involves providing a support having an evt protein or a representative fragment thereof; contacting the support with a binding partner that binds to the evt protein or representative fragment thereof; contacting the support with a candidate agent; and detecting the presence or absence of binding of the binding partner to the evt protein and thereby identifying the agent as one that modulates evt-mediated signal transduction.
Another embodiment of the invention concerns a knock-out mouse, wherein the wild-type evt-1 gene is replaced with a mutant evt-1 gene. Furthermore, a method for producing a genetically altered mouse that exhibits a defect in vesicle trafficking, signal transduction, G protein binding, or membrane biosynthesis and organization is an aspect of the invention. This method is practiced by providing an evectin gene targeting construct comprising an evt-1 gene having a modification resulting in an amino acid substitution; introducing said evt-1 gene having a modification resulting in an amino acid substitution and a selectable marker sequence into a mouse embryonic stem cell; introducing said mouse embryonic stem cell into a mouse embryo; transplanting said embryo into a pseudopregnant mouse; allowing said embryo to develop to term; identifying a genetically altered mouse whose genome comprises a modification of the evt-1 gene in both alleles; and breeding the genetically altered mouse of step (f) to obtain a genetically altered mouse whose genome comprises a modification of the endogenous evt-1 gene, wherein said disruption results in said mouse exhibiting a defect in vesicle trafficking, signal transduction, G protein binding, or membrane biosynthesis and organization.